Chemokines are secreted pro-inflammatory proteins of small dimensions (70-130 amino acids) mostly involved in the directional migration and activation of cells, especially the extravasation of leukocytes from the blood to tissue localizations needing the recruitment of these cells (Baggiolini M et al., 1997; Rossi D and Zlotnik A, 2000; Fernandez E J and Lolis E, 2002). Usually chemokines are produced at the site of an injury, inflammation, or other tissue alteration in a paracrine or autocrine fashion, triggering cell-type specific migration and activation.
Depending on the number and the position of the conserved cysteines in the sequence, chemokines are classified into C, CC, CXC and CX3C chemokines. Inside each of these families, chemokines can be further grouped according to the homology of the entire sequence, or of specific segments.
A series of heptahelical G-protein coupled membrane receptors, are the binding partners that allow chemokines to exert their biological activity on the target cells, which present specific combinations of receptors according to their state and/or type. An unified nomenclature for chemokine ligands and receptors, which were originally named by the scientists discovering them in a very heterogeneous manner, has been proposed to associate each of these molecule to a systemic name including a progressive number: CCL1 CCL2, etc. for CC chemokines; CCR1 CCR2, etc. for CC chemokines receptors, and so on.
The physiological effects of chemokines result from a complex and integrated system of concurrent interactions. The receptors often have overlapping ligand specificity, so that a single receptor can bind different chemokines, as well a single chemokine can bind different receptors. In particular, N-terminal domain of chemokines is involved in receptor binding and N-terminal processing can either activate chemokines or render chemokines completely inactive.
Even though there are potential drawbacks in using chemokines as therapeutic agents (tendency to aggregate, promiscuous binding), these molecules offer the possibility for therapeutic intervention in pathological conditions associated to such processes, in particular by inhibiting/antagonizing specific chemokines and their receptors at the scope to preventing the excessive recruitment and activation of cells, in particular leukocytes, for a variety of indications related to inflammatory and autoimmune diseases, cancers, and bacterial or viral infections (Carter P H, 2002; Schneider G P et al., 2001, Baggiolini M, 2001; Godessart N and Kunkel S L, 2001; Proudfoot A et al., 2000).
Amongst all the chemokines characterized so far, CC-chemokines, such as CCL5 (also known as RANTES; Appay V and Rowland-Jones S L, 2001) have been intensively studied to identify therapeutically useful molecules. Variants of CC-chemokines, missing up to nine N-terminal amino acids, have been tested for their activity as inhibitors or antagonists of the naturally occurring forms. These molecules are inactive on monocytes and are useful as receptor antagonists (Gong J and Clark-Lewis I, 1995; Gong J H et al., 1996; WO 99/16877). Alternatively, N-terminal extension of the mature CC-chemokine with one Methionine results in almost complete inactivation of the molecule, which also behaves as an antagonist for the authentic one (WO 96/17935).
Moreover, in order to perform structure-function analysis of CC-chemokines, variants containing substitutions or chemical modifications in different positions, as well as CC-chemokine derived peptides, have been tested for the interactions with receptors or other molecules, such as Glycosaminoglycans (GAGs). Some of these variants have been disclosed as having significatively altered binding properties, and sometimes they are active as CC-chemokine antagonists, having potential therapeutic applications in the treatment of HIV infection and inflammatory or allergic diseases (WO 99/33989; U.S. Pat. No. 6,057,123; PCT/EP01/11428; Nardese V et al., 2001; Martin L et al., 2001; Beck C et al., 2001; Hemmerich S et al., 1999).
However, none of these approaches have exhaustively studied the properties of all mutants deriving from the non-conservative substitutions in each single positions conserved in CC-chemokines, or in a subset of them.